Mutational analysis identifies a new functional domain of the thyroid hormone receptor.
We studied the functional significance of a region of the thyroid hormone receptor ligand binding domain which is conserved in all members of the erbA superfamily. The homologous region of the glucocorticoid receptor has been implicated in the binding of heat shock protein 90, and deletions of the glucocorticoid receptor that include this region result in constitutive activity. Both deletion and point mutations were made in this area of the rat beta 1 thyroid hormone receptor (amino acids 286-305), and the functional consequences of these mutations were analyzed in JEG cells by transient cotransfection along with a T3 responsive reporter gene. All mutations studied resulted in significant inhibition of ligand-dependent transcriptional regulation without inducing significant constitutive activity. For some mutations, the lack of transcriptional response correlated with a diminished ability to bind ligand. However, point mutations of amino acids 288, 290, and 300 resulted in impaired transcriptional regulation despite wild type T3 binding affinity. In addition, mutations of these three amino acids failed to impair localization of the receptor to the nucleus or binding to DNA in vitro. Cotransfection of plasmids expressing the wild type and mutant T3 receptor proteins resulted in inhibition of the wild type T3 receptor function. We conclude that this region of the rat beta 1 T3 receptor is essential for full transcriptional activity, but this is not due to a role in T3 binding, DNA binding, or nuclear localization. We postulate that nuclear factors may need to bind to this region for full transcriptional activity.
O'Donnell
A L
AL
Division of Endocrinology, University of Michigan Medical Center, Ann Arbor 48109.
Koenig
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D015870:Gene Expression / Q000187:drug effects; D008969:Molecular Sequence Data; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology
D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine